SUNY-ESF, Department of Environmental and Forest Biology, Syracuse, NY, United States of America.
PLoS One. 2019 Nov 14;14(11):e0222883. doi: 10.1371/journal.pone.0222883. eCollection 2019.
Bog turtles (Glyptemys muhlenbergii) are listed as Species of Greatest Conservation Need (SGCN) for wildlife action plans in every state it occurs and multi-state efforts are underway to better characterize extant populations and prioritize restoration efforts. However, traditional sampling methods can be ineffective due to the turtle's wetland habitat, small size, and burrowing nature. Molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying minute amounts of turtle DNA left behind in its environment (eDNA). Developing such methods for bog turtles has proved difficult partly because of the high sequence similarity between bog turtles and closely-related, cohabitating species, most often wood turtles (Glyptemys insculpta). Additionally, substrates containing bog turtle eDNA are often rich in organics or other substances that frequently inhibit both DNA extraction and qPCR amplification. Here, we describe the development and validation of a qPCR assay, BT3, targeting the mitochondrial cytochrome oxidase I gene that correctly identifies bog turtles with 100% specificity and sensitivity when tested on 201 blood samples collected from six species over a wide geographic range. We also developed a full-process internal control employing a genetically modified strain of Caenorhabditis elegans to improve DNA extraction methods, limit false negative results due to qPCR inhibition, and measure total DNA recovery from each sample. Using the internal control, we found that DNA recovery varied by over an order of magnitude between samples and likely explains the lack of bog turtle detection in some cases. Methods presented herein are highly-specific and may offer a more cost effective, non-invasive tool to supplement bog turtle population assessments in the Eastern United States. Poor or differential DNA recovery, which remains unmeasured in the vast majority of eDNA studies, significantly reduced the ability to detect bog turtle in their natural environment.
斑鳖（Glyptemys muhlenbergii）被列为每个州野生动物行动计划中最需要保护的物种（SGCN），并且正在进行多州合作，以更好地描述现存种群并优先进行恢复工作。然而，由于龟类的湿地栖息地、体型小和穴居的习性，传统的采样方法可能效果不佳。分子方法，如 qPCR，可以有效地量化龟类在其环境中遗留的微量 DNA，从而克服这一挑战。为斑鳖开发这种方法一直很困难，部分原因是斑鳖与亲缘关系密切、共同生活的物种（最常见的是木雕龟 Glyptemys insculpta）之间的序列高度相似。此外，含有斑鳖 eDNA 的基质通常富含有机物或其他物质，这些物质经常抑制 DNA 提取和 qPCR 扩增。在这里，我们描述了一种 qPCR 测定法 BT3 的开发和验证，该方法针对线粒体细胞色素氧化酶 I 基因，在对来自六个物种、分布广泛的 201 个血液样本进行测试时，具有 100%的特异性和灵敏度。我们还开发了一个完整过程的内部对照，使用经过基因改造的秀丽隐杆线虫（Caenorhabditis elegans）来改良 DNA 提取方法，减少由于 qPCR 抑制导致的假阴性结果，并测量每个样本的总 DNA 回收量。使用内部对照，我们发现 DNA 回收率在样本之间差异超过一个数量级，这可能解释了在某些情况下缺乏斑鳖检测的原因。本文介绍的方法具有高度特异性，可能为补充美国东部的斑鳖种群评估提供一种更具成本效益、非侵入性的工具。在绝大多数 eDNA 研究中，DNA 回收率差或差异较大，这大大降低了在其自然环境中检测斑鳖的能力。
